Sour cherries (P. cerasus), are one of the medicinal fruits used not only in fresh and frozen forms due to their significant antioxidant and polyphenol content but also in processed and commercial nutraceutical products due to their many uses. In this study, the total phenolic and flavonoid contents of sour cherry chloroform and methanol extracts and their inhibitory activities on pathogenic microorganisms and acetylcholinesterase enzyme were investigated. The methanol extract of P. cerasus fruit had higher total phenolic content, while the chloroform extract had higher flavonoid content. Extracts obtained from P. cerasus fruit significantly inhibited the growth of Candida albicans yeast and Escherichia coli and Staphylococcus aureus bacterial strains at low doses. The phenolic compound concentrations of the extracts were determined using liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Vanillic acid, naringenin, cichoric acid, and 4-hydroxybenzoic acid were the main constituents in the chloroform extract, whereas cichoric acid, citric acid, chlorogenic acid, and maleic acid were in the methanol extract. The chloroform extract showed higher acetylcholinesterase inhibitory activity compared to the methanol. The chemical structure, antipathogenic properties, and enzyme inhibitory ability of the extracts obtained from P. cerasus fruit suggest that they may be used in biomedical applications.